The overall goal of this research is to understand how transmitter-gated membrane channels (receptors) function and how their function is modified by pharmacological agents. These receptors allow cells in the brain to communicate, and their proper function is essential to normal brain function. They are also the targets for many drugs, both clinically used and recreational. This research focuses on a major class of human brain nicotinic receptors, the one composed of a4 and 2 subunits (a42 receptors). The functional receptor is composed of five subunits: two pairs of a4-2 subunits form the two acetylcholine-binding sites (ACh, the neurotransmitter which activates the receptor) while the 5th subunit does not contribute to an ACh-binding site. The a42 receptor has two forms, one in which the 5th subunit is an a4 subunit and the other in which it is a 2 subunit. The properties of the two forms differ physiologically and pharmacologically - for example when the 5th subunit is a4 the receptor has a low sensitivity to ACh, whereas when it is 2 the sensitivity to ACh is high. Accordingly, the proposed research will use techniques to define the subunit stoichiometry and arrangement so that the properties of a defined type of receptor will be studied. The first two aims of this proposal are to determine the mechanism (Aim 1) and site of action (Aim 2) for drugs which potentiate the activation of a42 receptors. Such drugs have been used or proposed for treatment of a number of disorders, including Alzheimer's disease. However, the site and mechanism of action of potentiating drugs are not known. Studies of function will emphasize analysis of receptor kinetics to provide novel information, while molecular studies will manipulate amino acids at specific subunit interfaces. The final aim will examine the structures in the a4 and 2 subunits which influence the ability of these subunits to assemble as the 5th subunit in the a42 receptor. Little is known about the structural basis of the rules for assembly of this receptor. Since the properties of the assembled receptor depend on its subunit composition, understanding the basis for selecting the 5th subunit is important for understanding how the overall properties of receptors in a neuron are affected by the palette of subunits expressed in the cell. The knowledge to be gained in the research will provide insights into how receptor function is modulated by chemicals, including clinically used drugs, and will increase our understanding of the mechanisms for the control of receptor function. Further, this work will provide new information on the physiological properties of the two forms of the a42 receptor and examine the mechanism for determining the choice of the 5th subunit. To accomplish these aims, receptors of known subunit composition and arrangement, and with mutations at specific interfaces, will be studied. PUBLIC HEALTH RELEVANCE: Receptors for neurotransmitters allow cells in the brain to communicate, and their proper function is essential to normal brain function. They are also the targets for many drugs, both clinically used and recreational. The goal of this research is to understand how these receptors function, how cells assemble receptors and how frequently used drugs act on receptors.